DOI: 10.1126/science.opms.p0600006

Life Science Technologies
Pharmacogenomics:
The Path Toward Personalized Medicine

Advances in pharmacogenomics, the scientific study of the relationship between individuals' genetic makeups and their responses to drugs, are bringing ever closer the goal of individualized treatments of diseases based on patients' genetic complements.

By Peter Gwynne and Gary Heebner

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In the half-dozen years since scientists sequenced the human genome, researchers have striven for the goal of personalized medicine: creating drugs explicitly designed to treat specific medical conditions in individual patients with the greatest efficiency and the fewest side effects. Several factors, including diet, age, and lifestyle, can influence human patients' responses to drugs. But researchers believe that an individual's genetic makeup has the greatest impact. To understand that genetic complement — and apply it to the development of personalized medicine — scientists rely on pharmacogenomics, the study of the ways in which a person's genetic makeup (DNA and RNA biomarkers) affects her or his response to drugs.

The field combines traditional pharmaceutical sciences such as biochemistry and organic chemistry with knowledge of genes, proteins, and single nucleotide polymorphisms (SNPs). Common techniques that specialists in pharmacogenomics use include DNA amplification, DNA sequencing, and gene expression analysis. "Tools and technologies are becoming increasingly sophisticated — everything from microarray methods to microfluidics to nanotechnology," says Amalia Issa, associate professor and director of the Program in Personalized Medicine and Targeted Therapeutics at the Abramson Center for the Future of Health of the University of Houston and The Methodist Hospital.

Those tools and technologies have helped to stimulate significant progress toward the Holy Grail of effective personalized medicine. "Our knowledge in the area that's relevant is moving along quite fast," says Ann Daly, professor of pharmacogenetics at the University of Newcastle upon Tyne, and editor-in-chief of Current Pharmacogenomics, published by Bentham Science Publishers.

The idea of tailoring treatments to patients isn't entirely new. "Physicians have taken note of items such as weight and age in diagnosis and prescription," explains Elliott Dawson, president and founder of BioVentures. What is new is the extension of those early steps toward personalized medicine to the link between genetic information and disease.

Established Links

Scientists have already established links for some diseases. "Clearly the area where we are advancing most rapidly is in oncology," says Patrice Milos, executive director of molecular profiling at Pfizer Global Research and Development. "What we used to look at pathologically can now be examined molecularly to better define the molecular events which have occurred in the tumor and therefore offer the potential opportunity to treat these diseases for our patients."

The approach already works in a limited way. "Herceptin [Genentech's drug for treating metastatic breast cancer] is a very special case, as we're tailoring the drug to the tumor, not really to the patient's genetic makeup," Daly explains. "There have also been some small-scale studies indicating that genetics may help us to improve the way in which we individualize the dose of the blood thinner Coumadin [also known as warfarin]." Another advance related to cancer involves the epidermal growth factor receptor, which has mutations in non—small cell lung cancer. "About 20 percent of lung cancers responded very quickly to new therapies from AstraZeneca," explains Andy Watson, senior director of market development for genetic analysis at Applied Biosystems. "It turns out that there were mutations in this gene that were very strongly associated with the identification of the 20 percent population that responded."

Despite those promising indications, Daly cautions against excessive expectations for movement toward personalized medicine on a broad scale. "The real issue is: How can we tailor drugs to treat the common chronic diseases?" she says. "That's moving along more slowly. At the moment, the hope is that current drugs can be used more effectively by targeting them to patients with particular genetic characteristics."

Achievement of that end would change the nature of medical treatment. Instead of one-size-fits-all blockbuster drugs, physicians would use pharmacogenomics-based diagnostics to determine which — and what dose — of a group of different drugs would best suit individual patients. This approach could enable patients to receive drugs that otherwise might not be prescribed, as they could have adverse reactions in some individuals. The ability to target patients with specific genetic complements could also resurrect treatments previously deemed too risky because they cause problematic side effects in too many patients. "Some players in industry are interested in the idea of rescuing stalled or dead drugs," Issa points out. "Gene Logic, for example, has a unit dedicated to drug positioning."

Druggable Targets

Current advances in pharmacogenomic tools and technologies promise advances in the move toward personalized medicine. "The first true advance is related to target discovery," says David Hoekzema, global director of pharmaceutical market development at Qiagen. "The most promising thing is the discovery of large numbers of druggable targets, such as nuclear receptors, kinases, and phosphatases. Those druggable targets have increased about 10-fold in the past couple of years." SNPs, the natural DNA variations that occur in individuals, also play a significant role. "There's been good evidence in correlating SNPs and their medical relevance," says Bill Marshall, executive vice president for research and operations and site manager at Dharmacon. David Smoller, vice president of R&D and operations for the biotechnology business unit at Sigma-Aldrich, identifies a further advance. "We're on the verge of a paradigm shift from discovering SNPs to developing biomarkers," he explains.

SNPs, nevertheless, provide a critical service, enabling researchers to develop a type of genetic fingerprint. Myriad Genetics, Perlegen Sciences, and Third Wave Technologies, among other companies, are working on methods for examining SNPs in human populations. BioVentures has developed what it calls DMG Diversity Navigators kits. These permit the detection of virtually all relevant genetic variants within a gene target by providing bidirectional sequence coverage for all the exons and flanking intron regions. "We use the kits at an early stage to identify variants of cytochromes," Dawson says. "We feel that the application is about two to five years away from being accepted in development."

BioVentures has developed some of its DMG Diversity Navigators products to cover both wild type and variant exons. Each contains optimized primers and buffers both for PCR amplification of the target sequences from genomic DNA samples and for sequencing the amplified template. "The kits save scientists time in developing reagents," Dawson explains. "We're also developing a series of products to address micro RNAs, which we expect to launch later this year for academic institutions and pharmaceutical companies."

Focus on Epigenetics

Researchers have also started to focus on epigenetics, the study of heritable changes in gene function that occur without a change in the nuclear DNA sequence. "It's now an emerging field that we see as very, very important," Qiagen's Hoekzema says. "The number of publications that have shown up in the field has gone up from about nine per year to roughly 200 in American journals of cancer research."

A major epigenetic mechanism in vertebrates, in addition to RNA-associated silencing and histone modification, is DNA methylation, which involves changes that can affect gene function. Traditionally, scientists studied methylation by digesting genomic DNA with methylation-sensitive restriction enzymes, followed by Southern blot analysis or PCR. Today, researchers can use more sophisticated and informative methods, including methylation-specific PCR (MSP) and sequencing.

Biosearch Technologies, Cambridge BioScience, and Imgenex as well as Qiagen offer reagents for this area of research. "We have tools for the analytical aspects of DNA preparation, which is really vital before you go into DNA methylation analysis," Hoekzema says. "We offer a number of products for the collection and stabilization and purification of DNA. We have a strategic alliance with Epigenomics to put together products and technologies that will provide the pretreatment and purification components already on the market with epigenomic DNA methylation technology, called Methylight. You then have a complete solution for collection, preparation and purification, and DNA methylation analysis."

Gene Expression and Silencing

Studies of gene expression also play a key role in the march toward personalized medicine. Such research involves the use of a variety of tools. To build a library of DNA clones, for example, researchers can either turn to companies that provide the tools to produce their own libraries or source large numbers of clones from companies such as ATCC, OriGene, and Promega. GE Healthcare, Invitrogen, and Stratagene, meanwhile, provide kits and reagents for gene expression studies. And scientists can use small interfering RNA to induce gene silencing in mammalian cells. Companies that offer kits and reagents for studies that rely on RNA interference (RNAi) include Ambion (now a part of Applied Biosystems), Dharmacon, Gene Therapy Systems, and Mirus.

Why is RNAi important in pharmacogenomics? "You can culture samples of prostate cancer in a dish, treat them with a suboptimal dose of a currently used chemotherapeutic agent, and come across with inhibitors of every gene in the genome," Dharmacon's Marshall explains. "You then look at where inhibition of the gene leads to enhancement of the agent's activity."

Dharmacon prides itself on the specificity of its RNAi tools. "The key factor in using RNAi effectively to develop pharmacogenomics tools is this specificity," Marshall explains.

Amplification Approaches

To increase throughput while decreasing human intervention in genomic research, vendors have developed a variety of laboratory automation tools, ranging from simple semiautomated liquid handling workstations to fully integrated robotic systems. Several companies offer instruments and reagents that allow scientists to process large numbers of samples in situations as diverse as simple chemical reactions and more complex ones such as PCR, whole genome amplification (WGA), SNP genotyping, and gene expression analysis.

Even in the early days of the Human Genome Project, scientists wanted to sequence more than selected regions. Investigators needed a way to sequence an entire genome — an achievement impossible with PCR alone. In the early 1990s, researchers developed several forms of whole-genome amplification. Ideally, these techniques generate multiple copies of every sequence in a sample of genomic DNA without disturbing the original copy. More DNA means that scientists can perform more downstream analysis. Companies that offer kits for WGA include GE Healthcare, Qiagen, and Sigma-Aldrich.

"Whole genome amplification allows researchers the ability to regenerate their precious research samples, providing unlimited analysis," Sigma-Aldrich's Smoller explains. "It works with samples that are in limited quantities, such as single cells. Other applications include amplification of nonrenewable samples from populations, such as blood and tissue samples from people whose samples are no longer available. It permits you to generate enough material to do your studies for the long haul." The technique also amplifies the entire genome with no bias for one sequence over another.

Using Sigma-Aldrich's GenomePlex WGA system, a researcher starts by chemically fragmenting the genome into random, overlapping short templates. The addition of universal primering sites turns these fragments into an OmniPlex library. Then, conventional PCR amplifies this library. The process starts with as little as subnanogram amounts of genomic DNA and produces 5–10 micrograms of amplified DNA in about three hours. By comparison, typical PCR requires about 100 nanograms of starting material. "The system is also suited for harshly treated samples, such as archived samples; it doesn't need perfect DNA," Smoller notes. "And it allows you to do single cell work." [GenomePlex and OminPlex are trademarks of Rubicon Genomics.]

Qiagen has its own technology for WGA. "Our REPLIg line of products is a series of systems that allow highly uniform DNA amplification across the entire genome," Hoekzema says. "We've developed it to perform on clinically relevant samples, such as blood and tissues. It's a clinical tool as much as a laboratory tool."

Secrets of Sequencing

DNA sequencing is among the techniques that scientists often use to analyze SNPs and genes. "It comes in at the discovery phase, identifying the particular mutations linked with disease," Applied Biosystems' Watson explains. "Finding them is the first problem, followed by getting the link to the particular disease or drug therapy."

DNA sequencing involves cutting DNA at precise locations and then separating the fragments by electrophoresis on agarose gels. Molecular biologists depend on a wide variety of restriction enzymes to cut DNA at different points along the strand so that data from the different digests can be compared in order to determine the actual DNA sequence of longer runs of DNA. Companies such as Invitrogen, New England Biolabs, Promega, and Roche Applied Science have played important roles in making these enzymes readily available to molecular biologists. "We have over 200 genes available from our website that customers can order as reagents to test against diseases," Watson says. "We have also designed over 16,000 genes for scientists who want to use our TargetSeqr to test a theory about genes that might be involved in a disease."

In addition, Applied Biosystems, LI-COR, and PerkinElmer offer automated DNA sequencers and other instruments for genomic research. Applied Biosystems' PRISM 3100 Genetic Analyzer and its advanced successor, the 3730 XL instrument, are multicolor fluorescence-based DNA analysis systems that use capillary electrophoresis with 16 capillaries operating in parallel. "The 3730 XL is modified to do very high throughput detection of mutations," Watson says.

This phase of pharmacogenomic research can also benefit from emerging life science technologies. Affymetrix, Illumina, NimbleGen, and SuperArray are among the companies that provide DNA microarrays for studies of genotyping and gene expression. The Affymetrix GeneChip Human Panel 1 10K cSNP kit includes a majority of the genes with validated (double-hit) nonsynonymous public SNPs that code for functional changes. And Caliper Life Sciences, among other firms, offers microfluidic devices for genomics research. Several companies developing microfluidic technologies have worked specifically in this line of products. Applied Biosystems, for example, has recently entered the field with its ABI Prism 7900HT Micro Fluidic Card for real-time gene expression research. This permits researchers to carry out many real-time PCR assays (and other assays) simultaneously on a single card.

Putting Pharmacogenomics into Practice

Beyond the lab, several companies and organizations have started to apply the results of pharmacogenomics research. Perlegen, for example, analyzes millions of SNPs in clinical trial participants to develop genetically targeted, late stage therapeutics and diagnostics. The company is actively licensing and developing its own portfolio of medicines and collaborating with partners such as Pfizer to position medicines more effectively in their portfolios and pipelines. Genentech has identified DNA biomarkers that predict the efficacy of Herceptin, its monoclonal antibody–based drug whose success in treating breast cancer has been linked to the presence of the HER-2 allele in patients with the disease. And Roche Molecular Diagnostics has developed the world's first pharmacogenomic microarray-based test approved for clinical use. The company's AmpliChip CYP450 Test provides comprehensive coverage of gene variations — including deletions and duplications — for the CYP2D6 and CYP2C19 genes, which play a major role in the metabolism of an estimated 25 percent of all prescription drugs. The company intends the test to be an aid for physicians in individualizing treatments and dosing for drugs metabolized through these genes.

At a broader level, Pfizer and Affymetrix joined with the U.S. National Institutes of Health and the Foundation for NIH in a partnership called the Genetic Association Information Network (GAIN) . Set up early this year, GAIN has the goal "of finding the multiple genetic factors that contribute to human disease," according to Pfizer's Milos. "The initiative is focused on making the data on the variety of diseases publicly available to help us all begin to understand the best targets to treat human disease. That translates into possible genetic pictures to pursue those targets and to offer better treatment for these diseases," she continues. "We're also very actively collecting genetic samples from clinical trial participants that allow us to do exploratory research on diseases and drug response."

Advances in pharmacogenomics plainly promise many benefits. For drug discovery and development, they imply an increase in the range of possible drug targets, decreases in the number of failed drug trials, and a reduction in the time it takes to get a drug approved. For patients this will mean fewer adverse drug reactions and decreases in the number of medications they will need to take to ensure the most effective therapy.

Peter Gwynne (pgwynne767{at}aol.com) is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner (gheebner{at}cell-associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

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